Iguratimod suppresses Tfh cell differentiation in primary Sjögren’s syndrome patients through inhibiting Akt/mTOR/STAT3 signaling

Background Iguratimod (IGU) reduces hypergammaglobulinemia and disease activity in pSS (primary Sjögren’s syndrome) patients. However, the therapeutical mechanism of IGU for pSS remains largely unknown. This study aimed to investigate the regulation of Tfh cell differentiation by IGU in pSS patients. Methods We prospectively enrolled 13 pSS patients treated with IGU for 3 months and examined circulating T cell and B cell subsets by flow cytometry. We measured Tfh cell differentiation treated by IGU in pSS patients and healthy controls. Transcriptome analysis combined with molecular docking were employed to identify potential therapeutical targets of IGU, which were verified by Western blot and Tfh cell differentiation. Results Tfh, plasmablast, and plasma cells were suppressed by IGU treatment at 1 and 3 months. Tfh cell differentiation and function were significant inhibited by IGU in pSS patients and healthy controls in vitro. Pyruvate dehydrogenase kinase 1 (PDK1) was identified as a target of IGU during Tfh cell differentiation, and the downstream Akt phosphorylation was attenuated by IGU. Moreover, the activity of mTORC1 and phosphorylation of STAT3 were suppressed by IGU, with downregulation of BCL6 and upregulation of PRDM1. Finally, Akt activator restored IGU-suppressed Tfh cell differentiation. Conclusions IGU suppresses Tfh cell differentiation in pSS patients through interacting with PDK1 and suppressing Akt-mTOR-STAT3 signaling. Supplementary Information The online version contains supplementary material available at 10.1186/s13075-023-03109-4.


Background
Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease featured with salivary, lacrimal, and other exocrine gland manifestations, with a high prevalence of 0.5-1.5% globally [1,2].Besides xerophthalmia and xerostomia, about half of pSS patients present hematologic, musculoskeletal, pulmonary, neurologic, and other systemic manifestations, which increase disease burden and mortality despite aggressive glucocorticoid and immunosuppressant therapy [3].
B cell overactivation is a hallmark of pSS, including polyclonal hypergammaglobulinemia composing rheumatoid factor (RF), antinuclear autoantibodies, and other autoantibodies, which are essential players for systemic manifestations [4].B cell-targeted therapies are tested in pSS [5].Ianalumab, an anti-BAFF receptor antibody, significantly improves disease activity and stimulates salivary flow and decreases peripheral B cells and immunoglobins [6,7].Telitacicept, a TACI-Ig targeting both BAFF and APRIL, dramatically improves disease activity and reduces immunoglobins and B cells in phase 2 trial [8].Remibrutinib, a BTK inhibitor, shows an improvement in disease activity in phase 2 study [9].Rituximab improves fatigue and serum immunoglobins [5].These encouraging data suggest that targeting B cells is a promising therapeutical approach for pSS.
Iguratimod (IGU) is a new DMARD approved for rheumatoid arthritis (RA) in China and Japan, with anti-inflammatory and immunomodulatory properties [10].We and others have reported that IGU significantly reduced immunoglobin and disease activity in pSS patients, which is a promising agent for pSS.IGU significantly decreases total B cells, BAFF-receptor + B cells, activated B cells, and plasma cells in pSS patients [11,12], suggesting IGU suppresses B cell differentiation.However, the therapeutical mechanism of IGU for pSS remains largely unknown.
In light of IGU suppressing B cell differentiation and Tfh cells regulating B cell differentiation, we hypothesized that IGU potentially suppressed B cell differentiation through repressing Tfh cells.In this study, we examined the Tfh cells in pSS patients before and after IGU treatment, and gained insights into the underlying mechanism using transcriptome analysis.

Subjects
We prospectively enrolled 13 pSS patients who fulfilled the classification criteria of the 2002 American European Consensus Group from Peking Union Medical College Hospital (PUMCH) (Supplementary Table S1).Patients received 25 mg twice daily IGU due to clinical manifestations and did not receive glucocorticoids and immunosuppressants.Clinical features, laboratory tests, and circulating Tfh, Th1, Th2, Th17, switched memory B, plasmablasts, and plasma cells were assessed at baseline, month 1, and month 3.The study was approved by the institutional review board of PUMCH (No. HS-1112).All participants provided written informed consent.

T cell proliferation, activation, and apoptosis assays
Naïve CD4 + T cells were labeled with 2 µM CFSE (BD Biosciences) for 15 min at 37 ℃, stimulated with anti-CD3 and anti-CD28, and were measured for proliferation using flow cytometry on day 3. T cell activation was assessed by staining with anti-CD69 at 16 h and anti-CD25 on day 3, and T cell apoptosis was examined on day 3 staining with Annexin-V and 7-AAD (BD Biosciences) for 15 min at room temperature.

Quantitative RT-PCR
Total RNA was extracted using TRIzol reagent (Thermo Fisher), was quantified by NanoDrop2000c spectrophotometer (NanoDrop Technologies), and was reverse transcribed into complementary DNA (cDNA) using PrimeScript RT Master Mix (Takara).Real-time PCR was performed using TB Green Premix Ex Taq II (Takara) and a Roche 480 II thermocycler, and relative expression against GAPDH was calculated using the comparative ΔΔCT method.The primer sequences are listed in Supplementary Table S2.

Transcriptional analysis of IGU-treated CD4 + T cells
Total RNA was collected from naive CD4 + T cells cultured under Tfh condition and was sequenced by Novogene (China).After RNA qualification and quantification, the library was prepared with quality control.Then, sequencing was initiated, followed by read mapping (hg19) and gene expression quantification.Differential expression was calculated with DESeq2 v.1.26.0.P-values were adjusted according to Benjamini and Hochberg's model.Differentially expressed genes were filtered based on adjusted p-value < 0.05 and |log2foldchange|> 1. Clus-terProfiler v.3.14.3 was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.

Target prediction and molecular docking
The molecular structure of IGU was obtained from PubChem and was searched in PharmMapper [25].The potential targets of IGU were sorted for duplicates and were translated into gene names.Discovery Studio Client v.4.5 was used to hydrogenate proteins, remove water, and remove ligand molecules.AutoDock v.4.2 was used to convert compound molecules and protein molecules into "pdbqt" format and finally run Vina for molecular docking.If binding energy was less than 0, the compound (ligand) and protein (receptor) bind spontaneously.A binding energy ≤ − 5.0 kcal/mol was considered as ligand bind to receptor stably.

Statistical analysis
Continuous variables were described as mean (standard deviation, SD) for normal distribution or median (interquartile range, IQR) for non-normal distribution, categorical variables were summarized as number (percentage).Comparisons between two groups were assessed using Student's t-test or Wilcoxon-Mann-Whitney test for continuous variables and Fisher's exact test for categorical variables as appropriate.Comparisons among three or more groups were assessed using analysis of variance (ANOVA) with Bonferroni adjusted p-value.A two-sided p-value < 0.05 was considered statistically significant.Data were analyzed using SPSS v.26.0 (IBM).

Transcriptome analysis of IGU-treated CD4 + T cells
We then conducted RNA-sequencing to elucidate the mechanism of IGU-inhibiting Tfh differentiation.IGU downregulated 275 genes and upregulated 126 genes (Fig. 3A) in CD4 + T cells, suggesting lower activation of T cells (Supplementary Figure S6A and B).The top 200 DEGs (up:100, down:100) showed distinct expression profiles of IGU-treated T cells (Fig. 3B).GO enrichment analysis revealed tubulin binding, chromosome segregation, and spindle were top enriched entities in molecular function, biological process, and cellular component, respectively (Supplementary Figure S11A-C).KEGG pathway enrichment analysis indicated 19 pathways were regulated by IGU, including the 2nd-ranked PI3K-Akt signaling pathway (Fig. 3C).We used target prediction based on pharmacophore mapping approach [25] to identify potential IGU-binding targets (Supplementary Table S3) and intersected with genes of enriched KEGG pathways (Supplementary Table S4, Supplementary Table S5).We identified that PI3K-Akt signaling pathway was the pathway with the most overlapping hits (Fig. 3D, Supplementary Table S6), including PDPK1, IL-2, JAK3, MET, INSR, RXRA, and EGFR.PDPK1, also known as pyruvate dehydrogenase kinase 1 (PDK1), was the top candidate of IGU binding according to Z score (Fig. 3E, Supplementary Table S6), which is a kinase phosphating AKT serine/threonine kinase 1 (Akt) in PI3K-Atk pathway.Finally, molecular docking validated the binding of IGU with PDK1 (Fig. 3F).Thus, transcriptome analysis implied that IGU regulated PI3K-Akt pathway through interaction with PDK1, which subsequently inhibited Tfh cell differentiation.

Discussion
In this study, we observed that IGU repressed Tfh cells as well as plasmablasts and plasma cells in pSS patients.Furthermore, we confirmed that IGU suppressed Tfh cell differentiation and B cell-helping function in vitro.Using transcriptome analysis combining molecular docking, we identified that IGU regulated PDK1 to inhibit PI3K-Akt signaling.Finally, we demonstrated that Akt-mTOR-STAT3 signaling was suppressed by IGU, resulting in downregulated BCL6 and upregulated PRMD1, and ultimately attenuated Tfh cell differentiation.
B cell hyperactivation in pSS is significantly suppressed by IGU, which is partially induced by attenuated activated B cells, plasma cell differentiation, and BAFF signaling.In RA patients, IGU attenuates B cell terminal differentiation through downregulating PKC signaling and transcription factor EGR1 [27].Tfh cells are essential for B cell activation, differentiation to plasma cells, antibody producing, antibody affinity maturation, and antibody-class switch [13], which plays pivotal role in autoantibody production of pSS, RA and lupus.In this study, we observed the IGU reduced Tfh cells, PD-1, ICOS, and IL-21 expressions in pSS patients, which are key molecules for B cell affinity maturation.Consistently, IGU treatment reduces serum IL-21 level in RA patients [28].In vitro, RA Tfh cells in PBMCs treated by IGU shows an impaired secretion of IL-21 [29].Thus, we proposed Tfh cells suppression as a novel mechanism of IGU treatment in pSS.IGU suppresses both B cell activation/differentiation and Tfh cell differentiation, which orchestrate to enhance B cell inhibition.Therefore, IGU is a promising immunomodulator for B cell overactivation and potential therapy targeting both B cells and Tfh cells in pSS.Given IGU suppresses Tfh cells in pSS and RA, IGU potentially suppresses Tfh cells in other Tfh-related autoimmune disease such as lupus [30,31].
The mechanism of Tfh regulation by IGU is not fully understood.IGU inhibits RA T cell glycolysis via suppressing Hif1α-HK2 signaling [29].We used transcriptome analysis to gain insights of signaling pathways regulated by IGU and combined analysis of potential IGU targets using molecular docking.Transcriptome analysis revealed that IGU suppressed PI3K-Akt signaling, but not NF-κB signaling, which is reported in synovial fibroblast-like cells [32].Furthermore, PDK1, a key kinase of PI3K-Akt signaling, was suggested to interact with IGU.PI3K-Akt signaling is an essential pathway for T cell response [26,33,34], which favors Tfh and Th17 differentiation [35,36].PI3K promotes PDK1 relocation to phospholipid-enriched membrane for phosphorylation and activated PDK1 phosphates Akt to transduct signaling [37] (Supplementary Figure S13).Since PDK1 promoted Tfh cell differentiation via AKT phosphorylation, mTORC1 activation, and STAT3 phosphorylation [26], we validated that IGU indeed inhibited PI3K-Akt signaling and downstream mTORC1 and STAT3 activation in Tfh cells, which was reversed by Akt activator.IGU also downregulated BCL6 and upregulated PRDM1, which are key transcription factors regulated by STAT3 [38] and synergically regulate Tfh genes including CXCR5, PD-1, and ICOS [39].Our data highlighted a new Akt-mTOR-STAT3 signaling regulated by IGU in Tfh differentiation and added a new layer of therapeutically mechanism of IGU on T cells.
Ours study have limitations.We observed IGU effect on Tfh cells in pSS patients up to 3 months, and the long-term effect remains unclear.We observed a stable reduction in serum immunoglobin in small number of pSS patients treated with IGU for up to 12 months (data not show), and we are following the enrolled patient for 6 months and beyond to determine the long-term effect of IGU on Tfh cells and B cells.Our study strongly suggested an interaction between IGU and PDK1; however, the direct binding domain of PDK1 by IGU and whether IGU interfered the PDK1 activation remain unclear.A selected mutation of potential PDK1 binding sites analysis might yield more informative insights of this molecular mechanism.

Conclusions
In summary, our study found that IGU suppresses Tfh cell differentiation in addition to B cell activation and differentiation.Mechanistically, IGU inhibits Akt-mTOR-STAT3 signaling by interacting with PDK1, subsequently represses BCL6 and upregulates PRDM1, and ultimately inhibits Tfh differentiation.Our results shed new light on that IGU modulates both Tfh cells and B cells in pSS and provide mechanical evidence on the therapeutical effect of IGU in pSS.